TRANSIENT GUS GENE-EXPRESSION IN BRASSICA-NAPUS ELECTROPORATED MICROSPORES

As a prerequisite to the transformation of rapeseed through androgenes is, we assessed the feasability of expressing foreign DNA in the male gametophyte of Brassica napus L. var. oleifera cv. Tapidor microspores by electroporation, using a square-wave-pulse discharge apparatus. Nu clease activities were observed only in young pollen grains and in the tapetum and disappeared after successive washings, The parameters of the electrical pulses used for the introduction of a fluorescent probe (calcein) into microspores without affecting microspore viability wer e determined in relation to different electroporation media. The optim al field conditions applied to microspores suspended in a BK-modified medium supplemented with 2% PEG were found to be 250 V/cm and 750 V/cm with respective pulse durations of 10 ms and 0.5 ms. Transient gene e xpression experiments using plasmids bearing the GUS reporter gene und er the control of CamV 35S or Zmg 13 promoters were used to test DNA t ransfer into microspores and pollen. Expression was significantly grea ter in electroporated microspores than in non-treated microspores. Thi s is the first step towards transgenic plants obtained by the haploid way.